© 2015, 2002 Elsevier Ltd. All rights reserved. Mycoplasmas and ureaplasmas are important human pathogens of adults and infants, predominantly affecting the respiratory and urogenital tracts. Being the smallest free-living organisms, they have evolved characteristics that make them unique among the prokaryotes and the focus of investigations aimed at learning what constitutes the minimal set of genes that can support independent life. Development of molecular-based methods for detection and characterization of these fascinating organisms over the past two decades has enabled more rapid and accurate diagnosis as well as increased knowledge about their basic biology and the infections they cause. Much information has been learned from genome sequencing and proteomics. Genome sequencing that took months to years a decade ago can now be done in a matter of hours for only a few percent of the investment once required. This chapter reviews the classification, structure, pathogenesis, detection, identification, and typing methods for Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum. Emergence and mechanisms involved in acquired antimicrobial resistance in these organisms is also discussed.